1. Field of the Invention
The present invention relates to a composition for the cryopreservation of cells with which an excellent survival rate of cells can be obtained.
2. Background Technology
Cryopreservation of cultured cells has been carried out for the purpose of preventing the degeneration of the cells by subculturing, preventing the contamination with unwanted bacteria during subculturing, and solving troubles in the maintenance of subcultures. For example, in the case of animal cells, conventionally, a preservation method for animal cells generally uses cryopreservation technology, in which cells are suspended in a culture solution containing 5-15% dimethyl sulfoxide (DMSO) or glycerin or bovine serum, the resulting suspension is sealed into tubes or ampoules for freezing and the sealed products are cooled rapidly at a rate of about −1° C./min using a programmed freezer and ultimately stored in liquid nitrogen (−196° C.).
However, when a culture solution is used for cryopreservation, the survival rate of frozen cells decreases so that it often takes time to grow the cells to a target number when they are thawed. On the other hand, when serum is used for cryopreservation, the cost for preservation increases in the case where various kinds of cell strains are frozen on a large scale since serum is quite expensive. Further, serum largely contains components which are primarily unnecessary for cell preservation, such as various kinds of cytokines, growth stimulating factors, hormones and ions, which likely deteriorates characteristics of preserved cells. Further, in the case where serum is used, or even where albumin purified from serum or plasma is used, problems such as the risk of viral infection still remain.
Furthermore, in the case where a programmed freezer is used, the procedure for cell freezing itself inevitably becomes complicated and the survival rate of the frozen cells problematically decreases as time lapses.
As a cryopreservation fluid with which the use of a programmed freezer is not necessary, for example, Japanese Patent Laid-open Publication No. H6-46840 (No. 1994-46840) has proposed a cryopreservation fluid with which the use of a programmed freezer is not necessary and direct transfer from an ice bath to an atmosphere at a temperature of −80° C. is possible. However, this cryopreservation fluid contains serum components.
Accordingly, a cryopreservation fluid for the preservation of cells with which the use of a programmed freezer is not necessary, and direct transfer from an ice bath to a freezer at −80° C. is possible and cells can be stored frozen over a long period of time without the need for a culture solution, serum, or components derived from serum has been desired.
Conventionally, amino acids, proteins, saccharides, alcohols, and the like are known as components to be used for a cryopreservation fluid for cells. For example, Shinozaki, K. et al.: FEBS Lett., 461(3), 205 (1999) and Takagi, H. et al.: Appl Environ Microbiol, 69(11), 6527 (2003) have reported that an amino acid, proline, has a cryoprotective effect. Further, Japanese Patent Laid-open Publication No. 2002-233356 has disclosed a cryopreservation fluid for cells with the use of saccharides (particularly glucose).
However, as far as the present inventors are aware, the cryoprotective effect of these components alone has not been sufficient as compared to serum components.
On the other hand, a silk protein, sericin, has been confirmed to have a cryoprotective effect on cells in Japanese Patent Laid-open Publication No. 2002-101869.